Preimplantation genetic testing for more than one genetic condition: clinical and ethical considerations and dilemmas.

STUDY QUESTION: Which clinical and ethical aspects of preimplantation genetic testing for monogenic disorders or structural rearrangements (PGT-M, PGT-SR) should be considered when accepting requests and counselling couples for PGT when applied for more than one condition (combination-PGT; cPGT-M/SR)? SUMMARY ANSWER: cPGT is a feasible extension of the practice of PGT-M/SR that may require adapting the criteria many countries have in place with regard to indications-setting for PGT-M/SR, while leading to complex choices that require timely counselling and information. WHAT IS KNOWN ALREADY: Although PGT-M/SR is usually performed to prevent transmission of one disorder, requests for PGT-M/SR for more than one condition (cPGT-M/SR) are becoming less exceptional. However, knowledge about implications for a responsible application of such treatments is lacking. STUDY DESIGN, SIZE, DURATION: Retrospective review of all (40) PGT-M/SR applications concerning more than one genetic condition over the period 1995-2018 in the files of the Dutch national PGT centre. This comprises all relevant national data since the start of PGT in the Netherlands. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Data regarding cPGT-M/SR cases were collected by means of reviewing medical files of couples applying for cPGT-M/SR. Ethical challenges arising with cPGT-M/SR were explored against the background of PGT-M/SR regulations in several European countries, as well as of relevant ESHRE-guidance regarding both indications-setting and transfer-decisions. MAIN RESULTS AND THE ROLE OF CHANCE: We report 40 couples applying for cPGT-M/SR of which 16 couples started their IVF treatment. Together they underwent 39 IVF cycles leading to the birth of five healthy children. Of the couples applying for cPGT, 45% differentiated between a primary and secondary condition in terms of perceived severity. In the light of an altered balance of benefits and drawbacks, we argue the 'high risk of a serious condition' standard that many countries uphold as governing indications-setting, should be lowered for secondary conditions in couples who already have an indication for PGT-M/SR. As a consequence of cPGT, professionals will more often be confronted with requests for transferring embryos known to be affected with a condition that they were tested for. In line with ESHRE guidance, such transfers may well be acceptable, on the condition of avoiding a high risk of a child with a seriously diminished quality of life. LIMITATIONS, REASONS FOR CAUTION: We are the first to give an overview of cPGT-M/SR treatments. Retrospective analysis was performed using national data, possibly not reflecting current trends worldwide. WIDER IMPLICATIONS OF THE FINDINGS: Our observations have led to recommendations for cPGT-M/SR that may add to centre policy making and to the formulation of professional guidelines. Given that the introduction of generic methods for genomic analysis in PGT will regularly yield incidental findings leading to transfer requests with these same challenges, the importance of our discussion exceeds the present discussion of cPGT. STUDY FUNDING/COMPETING INTEREST(S): The research for this publication was funded by the Dutch Organization for Health Research and Development (ZonMw), project number: 141111002 (Long term safety, quality and ethics of Preimplantation Genetic Diagnosis). None of the authors has any competing interests to declare.

BRCA1 mutation carriers have a lower number of mature oocytes after ovarian stimulation for IVF/PGD.

PURPOSE: The aim of this study was to determine whether BRCA1/2 mutation carriers produce fewer mature oocytes after ovarian stimulation for in vitro fertilization (IVF) with preimplantation genetic diagnosis (PGD), in comparison to a PGD control group. METHODS: A retrospective, international, multicenter cohort study was performed on data of first PGD cycles performed between January 2006 and September 2015. Data were extracted from medical files. The study was performed in one PGD center and three affiliated IVF centers in the Netherlands and one PGD center in Belgium. Exposed couples underwent PGD because of a pathogenic BRCA1/2 mutation, controls for other monogenic conditions. Only couples treated in a long gonadotropin-releasing hormone (GnRH) agonist-suppressive protocol, stimulated with at least 150 IU follicle stimulating hormone (FSH), were included. Women suspected to have a diminished ovarian reserve status due to chemotherapy, auto-immune disorders, or genetic conditions (other than BRCA1/2 mutations) were excluded. A total of 106 BRCA1/2 mutation carriers underwent PGD in this period, of which 43 (20 BRCA1 and 23 BRCA2 mutation carriers) met the inclusion criteria. They were compared to 174 controls selected by frequency matching. RESULTS: Thirty-eight BRCA1/2 mutation carriers (18 BRCA1 and 20 BRCA2 mutation carriers) and 154 controls proceeded to oocyte pickup. The median number of mature oocytes was 7.0 (interquartile range (IQR) 4.0-9.0) in the BRCA group as a whole, 6.5 (IQR 4.0-8.0) in BRCA1 mutation carriers, 7.5 (IQR 5.5-9.0) in BRCA2 mutation carriers, and 8.0 (IQR 6.0-11.0) in controls. Multiple linear regression analysis with the number of mature oocytes as a dependent variable and adjustment for treatment center, female age, female body mass index (BMI), type of gonadotropin used, and the total dose of gonadotropins administered revealed a significantly lower yield of mature oocytes in the BRCA group as compared to controls (p = 0.04). This finding could be fully accounted for by the BRCA1 subgroup (BRCA1 mutation carriers versus controls p = 0.02, BRCA2 mutation carriers versus controls p = 0.50). CONCLUSIONS: Ovarian response to stimulation, expressed as the number of mature oocytes, was reduced in BRCA1 but not in BRCA2 mutation carriers. Although oocyte yield was in correspondence to a normal response in all subgroups, this finding points to a possible negative influence of the BRCA1 gene on ovarian reserve.

Mutations in the vasopressin type 2 receptor gene (AVPR2) associated with nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus (DIR) is an X-linked disorder characterized by insensitivity of the distal nephron for the pituitary hormone, vasopressin. The genetic map location of the DIR gene on chromosome Xq28 coincides with the physical map location of the functional vasopressin renal V2-type receptor. Recently, the human and rat cDNAs for the vasopressin V2 receptor (AVPR2) have been identified. We show here that the structural AVPR2 gene is localized between DXS52 and G6PD, which is within the genetic map location of DIR. We also tested eight X-linked DIR probands and their families for mutations in one of the most conserved extracellular regions of AVPR2: in three of them, we have identified point mutations resulting in non-conservative amino acid substitutions which cosegregated with DIR in all families.

Validation of preimplantation genetic diagnosis by PCR analysis: genotype comparison of the blastomere and corresponding embryo, implications for clinical practice.

The aim of this study was to validate the overall preimplantation genetic diagnosis (PGD)-PCR procedure and to determine the diagnostic value. Genotyped embryos not selected for embryo transfer (ET) and unsuitable for cryopreservation after PGD were used for confirmatory analysis. The PGD genotyped blastomeres and corresponding embryos were compared, and morphology was scored on Day 4 post fertilization. To establish the validity of the PGD-PCR procedure and the diagnostic value, misdiagnosis rate, false-negative rate and negative predictive value were calculated. Moreover, comparison on the validity was made for the biopsy of one or two blastomeres. For the total embryo group (n = 422), a misdiagnosis rate of 7.1% and a false-negative rate of 3.1% were found. The negative predictive value was 96.1%. Poor morphology Day 4 embryos (Class 1) were over-represented in the embryo group in which the blastomere genotype was not confirmed by the whole embryo genotype. The misdiagnosis rate of Class 1 embryos was 12.5% and the false-negative rate 17.1%. Exclusion of these embryos resulted in a misdiagnosis rate of 6.1%, a false-negative rate of 0.5% and a negative predictive value of 99.3%. The two blastomere biopsies revealed a significant higher positive predictive value, lowering the misdiagnosis rate, whereas the negative predictive value remained the same. In conclusion, the PGD-PCR procedure is a valid diagnostic method to select unaffected embryos for ET. The misdiagnosis and false-negative rates decrease by rejecting Class 1 embryos for ET. The biopsy of a second blastomere improves the positive predictive value, lowering the misdiagnosis rate.

Genotype assignment of haemophilia A by use of intragenic and extragenic restriction fragment length polymorphisms.

We performed DNA analysis in 20 families with haemophilia A in order to evaluate its usefulness for carrier detection and prenatal diagnosis. The polymorphic BclI site within intron 18 of the factor VIII gene and the extragenic TaqI and BglII polymorphic sites which are detected by the random DNA probes designated St14 and DX13, respectively, were investigated for. Two events of recombination were found between the St14 and the haemophilia A locus in 51 informative meioses. In one of these recombinant meioses crossing over had also occurred between the DX13 and the haemophilia A locus. No further crossovers between the DX13 and the haemophilia A locus were found in 20 informative meioses. Segregation analysis of the polymorphic markers and the deleterious mutation within the families allowed a diagnosis at the gene level for 52 out of 57 potential carriers. The new method considerably decreased the uncertainty about carriership for seventeen of the nineteen women with a probability of carriership between 5% and 95% based on pedigree analysis and factor VIII assays. In seven cases chromosome and DNA analysis of a chorionic villus biopsy was carried out. Three of the fetuses were female, four were male. Three of the male fetuses had inherited the normal maternal X-chromosome and were, therefore, not affected. For another male fetus no diagnosis at the gene level was possible since the mother was homozygous for all the known restriction fragment length polymorphisms within or closely linked with the haemophilia A locus.

High prevalence of the fra(X) syndrome cannot be explained by a high mutation rate.

The overall prevalence of the fragile X [fra(X)] mutation, as determined by population studies, is approximately 1 in 850 [Gustavson et al., 1986; Webb et al., 1986]. This prevalence suggests a very high mutation rate which, in turn, suggests that many patients have to represent sporadic cases. In order to obtain an accurate estimate of the proportion of sporadic cases, we performed genealogic, cytogenetic and DNA linkage studies as well as direct analysis of the CGG repeat in relatives of 84 fra(X) probands. We did not find any evidence for the presence of sporadic cases. In 11 probands consanguinity could be proven by the detection of common ancestors, in 5 related families up to 9 generations ago. In the other 6 related families the mutation could be traced back 4-6 generations. In 3 or more generation families we were able to demonstrate that half of the probands carried the grandpaternal fra(X) gene. These results imply that rather than a high mutation rate, both Normal Transmitting Males (NTM's) and mentally normal female carriers contribute considerably to the high prevalence of the fra(X) syndrome.

Prediction of mental status in carriers of the fragile X mutation using CGG repeat length.

For genetic counseling in fragile X (fra(X)) families, it is important to know the diagnostic impact of the CGG repeat length in carriers of the fragile X mutation. We have analyzed the CGG repeat length in 106 males and 73 females who had inherited the maternal fra(X) mutation. The sensitivity, specificity, and predictive value of the CGG repeat analyses, as measured on Southern blots of PstI/EcoRI digests, were calculated. In males the sensitivity, specificity and negative predictive value were 99%, 100% and 94%; respectively. In females the specificity (60%) and, consequently, the positive predictive value (82%) was reduced. Therefore, it remains impossible to predict accurately whether a female fetus demonstrating a full mutation will be affected. On the other hand, the negative predictive value of the CGG test in females was 100%. We have no evidence, as yet, that for female carriers with the full mutation, cytogenetic analysis is of additional value to predict the mental status. However, we have observed one mentally retarded fra(X) case with extreme mosaicism in which a typical premutation fragment was the predominant DNA species. Therefore, until more data and better DNA tests are available, we would like to advocate additional cytogenetic investigation if in a fetus a CGG repeat length in the premutation range is found.

Parental origin of the fra(X) gene is a major determinant of the cytogenetic expression and the CGG repeat length in female carriers.

The fragile X [fra(X)] syndrome is the most frequent form of inherited mental retardation, and co-segregates with a fragile site at Xq27.3 as well as with insertion of a variable number of trinucleotide repeats in the 5'-end of the FMR-1 gene. As the fra(X) gene is transmitted by females as well as males, we have investigated whether the parental origin of the fra(X) gene has an effect upon the cytogenetic expression and CGG repeat length. An increased fragment length of 0.2-0.6 kb appeared to be associated with a very low expression or even complete absence of the fragile site as well as with a normal phenotype, and was seen mostly in cases of paternal inheritance. However, in most female carriers with the maternally inherited fra(X) gene we found dispersed fragments ranging from 1.4-6.5 kb or even complete absence of a hybridization signal. Within the group of female carriers with the maternally inherited fra(X) gene we found a statistically significant correlation between the level of the cytogenetic expression and the PstI restriction fragment length encompassing the CGG repeat. These data can be taken as indirect evidence that CGG repeat length and cytogenetic expression are causally related.

Validation of linkage-based DNA-diagnosis of fragile X gene carriers with the CGG repeat probe.

We have evaluated our carrier testing for the fragile X [fra(X)] syndrome, which was based on linked DNA markers, with the direct analysis of the CGG repeat sequence in the fra(X) gene. PstI and EcoRI blots were hybridized with a probe derived from the region just 3' of the CGG repeat in Xq27.3. We found the mutation analysis to be very sensitive as all 71 obligate gene carriers as well as 135 fra(X) patients tested showed evidence for an increased restriction fragment length encompassing the CGG repeat sequence with or without dispersion of the hybridization signal (mosaicism). Based on linked DNA markers, 6 out of 50 cytogenetic negative and mentally normal males at risk and 15 of 72 females at risk had inherited the allele at risk. All of these diagnoses could be confirmed by analysis of the CGG repeat length.

Penetrance of fra(X) gene: influence of grandparental origin of the gene, mental status of the carrier mother, and presence of a normal transmitting male.

Previous studies have indicated that the fragile X [fra(X)] gene does not show full penetrance (mental impairment) in carrier females or "carrier" males. The phenomenon of non-expressing carrier males distinguishes the fra(X) syndrome from all other known X-linked disorders. Moreover, penetrance of the fra(X) gene apparently does not show random distribution within fra(X) families, but seems to be reduced in sibs of normal transmitting males (NTM's). The availability of many large multigeneration fra(X) families, studied by cytogenetic and DNA analyses, enabled us to refine the estimates of the penetrance. From our data we conclude that the penetrance in daughters of carrier females is determined by the mental status of the mother. In sons of carrier females, the observed penetrance appears to be influenced by the grandparental origin of the gene as well as by the mental status of the mother. However, in contrast with the average penetrance, we observed a strongly reduced penetrance of the fra(X) gene in brothers (14%) and sisters (21%) of NTM's. These findings have profound implications for genetic counseling.

Multipoint linkage analysis of DXS369 and DXS304 in fragile X families.

Diagnosis of carriers of the fragile-X mental retardation gene is hampered by the paucity of tightly linked DNA markers. Recently, 2 new DNA markers RN1 (DXS369) and U6.2 (DXS304) have become available. Both markers are tightly linked to the fragile-X locus, but their location relative to the fragile site was not known with certainty. We have tested these new markers in a multipoint linkage analysis of 26 fragile-X families typed for DXS105 as a proximal marker and DXS52 as a distal marker. Our results establish the order DXS105-DXS369-fra(X)-DXS304-DXS52, which is in agreement with physical mapping results.

Photon absorbed fractions for cylindrical geometry: homogeneous nonradioactive cylinder containing a homogeneous cylindrical distribution of activity.

The purpose of this study was to calculate photon absorbed fractions in tissue surrounding a radioactive source where both the source and the surrounding tissue are assumed to have cylindrical geometry. Specifically, we treated two cases: the case of a cylindrical source of homogeneous activity placed in air, and second, the case of a cylindrical source of homogeneous activity placed in a cylindrical nonradioactive absorbing material. In this study we offer an analytical solution to these problems followed by numerical integration. The computer program allowed for very general calculations, e.g., different tissues, different geometrical setups. Tables of absorbed fractions have been developed for commonly used radionuclide energies and tissue-equivalent material. A comparison between our results and the results of other related studies showed the advantages and limitations of this approach.

Photon-absorbed fractions for cylindrical geometry: a TLD photon-absorbed fraction model.

The purpose of this study is to develop a mathematical model and calculate photon-absorbed fractions in a homogeneous nonradioactive cylinder placed inside off-center and outside a cylindrical homogeneous distribution of activity. In the second case, both the radioactive cylinder and the nonradioactive one are placed in a tissue-equivalent nonradioactive medium. The values of the photon-absorbed fractions are investigated for various geometrical configurations using water as the material filling the cylinders and the medium in between and an isotope commonly used in Nuclear Medicine, 99mTc. The calculations for off-center cylinders allows for modeling inhomogeneous distributions of activity within a tumor by placing several "cold" cylinders of various sizes in a radioactive finite cylinder. This three-dimensional model calculates photon-absorbed fractions for inhomogeneous activity distributions that can be used in quantitative nuclear medicine for self-absorption correction, thus introducing a more realistic correction than the one-dimensional corrections. These calculations are also used to model the response of a cylindrical TLD (thermoluminiscent dosimeter) placed inside a homogeneous radioactive cylinder and outside the homogeneous radioactive cylinder, in an absorbing nonradioactive surrounding medium. The purpose of these calculations is to evaluate the photon-absorbed fraction in the TLD as an instrument of measuring the time-integrated activity of a homogeneous radioactive source versus an inhomogeneous one. The dependence of the TLD-absorbed fraction on the position of the TLD with respect to the radioactive cylinder is investigated.

[Genetic diagnosis of IVF embryos: preliminary results from 'preimplantation genetic diagnoses' in the Netherlands]. / Genetische diagnostiek bij IVF-embryo's: eerste ervaringen met 'preïmplantatiegenetische diagnostiek' in Nederland.

Preimplantation genetic diagnosis (PGD) is a very early form of genetic testing. It involves testing one or two cells taken from a recent embryo of eight cells produced by in vitro fertilization, and selective transfer of genetically normal embryos. So far in the Academic Hospital Maastricht, the Netherlands, 20 couples have undergone PGD, resulting in 6 ongoing pregnancies (one twin pregnancy). In three women the indications for PGD were: cystic fibrosis, sex-linked Pelizaeus-Merzbacher disease and chromosomal translocation, respectively. In the Netherlands PGD is only allowed if there is a high risk of a serious genetic disease. PGD can be carried out in Maastricht for: cystic fibrosis, sex-linked diseases, chromosomal abnormalities, fragile X syndrome, spinal muscular atrophy and myotonic dystrophy. The advantage of PGD is that it excludes the necessity of a therapeutic abortion. Disadvantages ages are the requirement of in vitro fertilization, which has only a 15-20% pregnancy rate, and the experimental nature of the PGD procedure. To date, about 200 children have been born worldwide following PGD.

[Results from 10 years of preimplantation-genetic diagnostics in The Netherlands]. / Resultaten van 10 jaar preimplantatiegenetische diagnostiek in Nederland.

OBJECTIVE: To report the data from couples who were referred for preimplantation-genetic diagnostics (PGD) and treatment due to a significantly increased risk of offspring with a serious genetic disorder. DESIGN: Descriptive, prospective. METHOD: Data were collected from couples that underwent PGD in the period 1993/'03 at Maastricht University Hospital. Embryos produced by means of in-vitro fertilisation (IVF) were subjected to genetic tests several days after fertilisation. Subsequently 1 or 2 unaffected embryos were transferred to the uterus. Where there was an increased risk of a male with an X-linked genetic disorder, the gender was determined using fluorescence in-situ hybridisation (FISH). This method was also used to detect structural chromosomal abnormalities. The polymerase chain reaction (PCR) method was used for mutation detection and/or marker analysis of monogenetic disorders. RESULTS: A total of 691 couples were referred for PGD. The most frequent indications were X-linked disorders (30%), in particular Fragile-X syndrome, Duchenne/Becker muscular dystrophy and haemophilia A/B. This was followed by autosomal dominant disorders (26%), such as Huntington's disease and myotonic dystrophy, and then structural chromosomal abnormalities (24%). A total of 120 women underwent 260 PGD cycles. An embryo transfer was possible in 158 of the cycles and this resulted in 45 successful pregnancies. The pregnancy rate was 17% per cycle initiated and 28% per cycle with embryo transfer. Up until december 2003 29 singletons, 8 sets of twins and 1 set of triplets were born. There were no misdiagnoses and none of the babies had congenital abnormalities. CONCLUSION: PGD was a reliable and successful method, with pregnancy rates similar to those of IVF or intracytoplasmatic sperm injection. PGD should be stated as an alternative during the preconception counselling of couples with an increased genetic risk, especially for disorders where PGD can be routinely applied, such as Huntington's disease, myotonic dystrophy, cystic fibrosis, spinal muscular atrophy, Fragile-X syndrome and structural chromosomal abnormalities.

Molecular cloning and restriction endonuclease mapping of the rat cytomegalovirus genome.

Rat cytomegalovirus (RCMV) DNA was cleaved by restriction endonuclease EcoRI into 24 fragments ranging in mol. wt. from 34 X 10(6) to 0.20 X 10(6), of which 18 fragments could be cloned in plasmid pACYC 184. Restriction endonuclease XbaI cleaved the RCMV genome into 28 fragments, ranging in size from 44 X 10(6) to 0.81 X 10(6), of which 24 fragments were cloned in plasmid pSP62-PL. Among the restriction fragments that could not be cloned were two major terminal colinear fragments, EcoRI-A (34 X 10(6)) and XbaI-A (44 X 10(6)). Thus, the complete sets of recombinant plasmids spanned about 70% of the RCMV genome. Our mapping results including determination of the termini of the genome, characterization of double digestion products of restriction fragments and cross-hybridization of 35S-labelled (cloned) EcoRI and XbaI fragments to Southern blots of EcoRI-, XbaI- or BglII-cleaved RCMV DNA, allowed us to construct the EcoRI and XbaI restriction maps of RCMV DNA. Since no cross-hybridization between internal fragments was seen, it is concluded that the RCMV genome consists of a long unique sequence of 224 kilobases without internal inverted repeat sequences, which is similar to the structures of murine and guinea-pig CMV DNA but unlike that of human CMV DNA. In a minor population (approx. 20%) of the RCMV DNA, one terminus was found to be larger by 0.35 X 10(6) mol. wt. The nature of this fragment is unclear at the moment.